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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
  • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate

Definitions

• This invention relates to immobilization of proteases produced by bacteria belonging to genus Streptomyces. More particularly, this invention relates to a water-insoluble, enzymatically active immobilized protease composition which comprises non-specific proteases having esterase activities and amidase activities produced by bacteria belonging to the genus Streptomyces immobilized on an anion exchange polysaccharide having a total anion-exchange capacity of not less than 0.5 meq/g or a water-insoluble macroporous, highporous or macroreticulated anion-exchange resin having specific surface area of not less than 1 m 2 /g and a total anion-exchange capacity of not less than 0.5 meq/g, and also to a process for producing the same.
• Enzymes are generally known to have substrate specificity, namely to have catalytic effect on specific substrates which are different depending on respective enzymes.
• bacteria belonging to the genus Streptomyces e.g. Streptomyces griseus, Streptomyces fradiae, Streptomyces erythreus, Streptomyces rimosus and Streptomyces flavovirens are known to have a broad substrate specificity, namely to produce so called non-specific proteases.
• Such a broad specificity is due to the fact that bacteria belonging to the genus Streptomyces produce simultaneously several kinds of endoproteases and several kinds of exoproteases which are relatively similar in physical and chemical properties to one another.
• proteases obtained from Streptomyces griseus, Streptomyces fradiae and Streptomyces and erythreus contain both endoproteases having substrate specificity such as the type of trypsin like activity, the type of chymotrypsin like activity, and the type of elastase like activity and exoproteases having substrate specificity such as the type of carboxypeptidase like activity and the type of aminopeptidase like activity.
• the type of trypsin like activity herein refers to protease having specificity on cationic amino acid residue such as of lysine or arginine; the type of chymotrypsin like activity to protease having specificity on large hydrophobic amino acid residue such as of tryptophan, tyrosine or phenyl alanine; and the type of elastase like activity to protease having specificity on small amino acid residue such as of glycine or alanine.
• non-specific proteases proteases produced by bacteria belonging to the genus Streptomyces can act on various substrates and therefore they are of a great practical value.
• these proteases have potent esterase and amidase activities not only on esters or amides of amino acids but also esters or amides of carboxylic acids in general.
• their catalytic effect is characterized by advantageous selective reactivity of an enzyme such that they act preferentially on L-isomer with little or no reaction with D-isomer.
• non-specific proteases produced by bacteria belonging to the genus Streptomyces are expected to be effective for optical resolution of esters or amides of amino acids or carboxylic acids having asymmetric carbons or introduction or chirality into prochiral molecules.
• proteases obtained from Streptomyces Sp. have many practical advantages such that they can be produced more cheaply and easily in a greater amount than proteases produced by animals.
• an enzyme reaction is conducted by dissolving an enzyme in water thereby to allow the enzyme to act on a substrate in an aqueous solution.
• the fact that the enzyme reaction is a homogeneous reaction in an aqueous solution is a great hindrance to performance of continuous reaction in industrial applications and also makes it very difficult to recover remaining active enzymes for repeated use after the reaction.
• complicated operational procedures are necessary for separation and purification of the reaction product which is present in admixture with enzymes. From these standpoints, it is practically very valuable to immobilize the non-specific proteases thereby to make them water-insoluble.
• proteases obtained from bovine kidney and spleen have been adsorbed on diethylaminoethyl cellulose.
• the resultant crude proteases were extremely low in activity.
• a crude enzyme solution which is low in specific activity and obtained just after removal by filtration of cells from a culture broth or after being merely subjected to preliminary purification procedure such as organic solvent precipitation method, can be mixed with an anion exchange polysaccharide having a total anion-exchange capacity of not less than 0.5 meq/g or a macroporous, highporous or macroreticulated anion-exchange resin having a total anion exchange capacity of not less than 0.5 meq/g and a specific surface area of not less than 1 m 2 /g to be readily immobilized thereon by adsorption; that the crude enzyme is partially purified through the immobilization procedure; that the immobilized enzyme is surprisingly increased by two to several times or more in esterase activities of the types of trypsin like activity, chymotrypsin like activity and elastase like activity and
• An object of the present invention is to provide a water-insoluble, enzymatically active enzyme composition which is obtained by adsorbing non-specific proteases having esterase activities and amidase activities produced by bacteria belonging to the genus Streptomyces on an anion-exchange polysaccharide having a total anion-exchange capacity of not less than 0.5 meq/g or a macroporous, high-porous or macroreticulated anion-exchange resin having a specific surface area of not less than 1 m 2 /g and a total anion-exchange capacity of not less than 0.5 meq/g.
• the other object of the present invention is to provide a method for immobilizing the aforesaid crude proteases simultaneously with purification thereof by treating said proteases with the anion-exchanger or anion-exchange resins as described above.
• the anion-exchange polysaccharide to be used in the present invention may include anion-exchange celluloses such as diethylaminoethyl cellulose (DEAE-cellulose), triethylaminoethyl cellulose (TEAE-cellulose), guanidoethyl cellulose (GE-cellulose) and the like; and anion-exchange Sephadex such as diethylaminoethyl Sephadex (DEAE-Sephadex), diethyl-2-hydroxylpropylaminoethyl Sephadex (QAE-Sephadex) and the like. Among them, DEAE-Sephadex and QAE-Sephadex are particularly preferred. "Sephadex” refers herein to trade names of cross-linked dextran (Pharmacia Fine Chemical Co.).
• anion-exchange resin having a specific surface area of not less than 1 m 2 /g and a total anion-exchange capacity of not less than 0.5 meq/g which is an immobilization carrier of the present invention
• ion-exchange resins or adsorbent resins known under the trade marks such as Amberlite, Dowex, Duorite and Diaion, those which have anion-exchange groups and are commercially available macroporous, highporous or macroreticulated anion-exchange resins with large specific surface area and porosity which are often called as MR (macroreticular) type, MP (macroporous) type, macronetwork structure type or highporous type.
• the anion-exchange resins may alternatively be prepared by various conventional methods.
• a macroporous resin can be obtained by permitting a compound inert to polymerization to be present in a polymerization system when styrene is copolymerized with suitable amount of divinyl benzene to prepare an ion-exchange resin matrix and after the polymerization, removing said compound by extraction with a solvent from the resulting resins obtained.
• This resin matrix is then subjected to chloromethylation of the resin matrix, followed by amination by conventional method, whereby a macroporous resin having anion-exchange groups can be obtained.
• the water-insoluble enzymatically active composition of the present invention can be prepared by first treating ion-exchangers by conventional method with an aqueous hydrochloric acid solution (0.01 to 3 M conc., preferably 0.05 to 1 M conc.) or an aqueous caustic soda solution (0.01 to 3 M conc., preferably 0.05 to 1 M) to activate ion-exchange groups or making the resins buffered at pH 5.5 to pH 8.5 with a suitable buffer solution (0.01 to 3 M conc., preferably 0.05 to 1 M) having buffer action within the range between pH 5.5 and pH 8.5 such as a phosphate buffer solution or a borate buffer solution, then immersing the thus treated anion-exchanger in a buffered enzyme solution for sufficient time, followed by stirring, if desired, and thereafter recovering the anion-exchanger, followed by filtration and washing.
• an aqueous hydrochloric acid solution (0.01 to 3 M conc., preferably 0.05 to
• the immobilization by adsorption is carried out at a temperature of 40° C. or lower, more preferably around 4° C.
• the adsorption time is desirably two hours or longer.
• the thus obtained water-insoluble proteases have high esterase, amidase and peptidase activities and are stable, provided that they are not washed with saltous solutions having high ionic strength. If desired, they may be dried by such a method as lyophilization so as to be storable for a long time and convenient for transportation.
• the said enzymes can be adsorbed in an amount of 400 mg per one gram of the anion-exchanger at its maximum, depending on the kinds, the specific surface area and porosity of the anion-exchanger employed.
• Example 1 was repeated, except that Amberlite IRA-93 (ion-exchange capacity: 4.6 meq/g) was used in place of the DEAE-Sephadex and the pH at the time of immobilization and measurement was changed from 8.0 to 7.2, to carry out similar immobilization procedure and activity measurement.
• the amount of immobilized enzyme was found to be 34 mg and the enzyme activity in a solution before immobilization, which was 6.5 ⁇ moles/mg.hr to BAEE at pH 7.2, was elevated by immobilization to 32 ⁇ moles/mg.hr.
• the activity on ATEE at pH 7.2 and 30° C. was found to be 7.3 ⁇ moles/mg.hr for enzyme solution before immobilization and 19 ⁇ moles/mg.hr for immobilized enzyme.
• the activity on 7th day after immobilization was 18 ⁇ moles/mg.hr.
• Immobilization and activity measurement were conducted under the same conditions as in Example 4 except that the immobilization carrier was changed from QAE-Sephadex to Duorite A 161 (ion-exchange capacity: 3.5 meq/g).
• the amount of immobilized enzyme was 64 mg and the activity of the immobilized enzyme 16.5 ⁇ moles/mg.hr.
• Example 1 For evaluation of the elastase type activity of the crude enzyme used in Example 1, there was measured activity on N-benzoyl-DL-alanine methyl ester (BAlME) which is a specific substrate for elastase. Before the immobilization, no activity was observed at the reaction temperature 40° C. at pH of 7.1 and 8.0. Then, 52 mg of the crude enzyme was immobilized by adsorption on 1 g of DEAE-Sephadex A-25 in a phosphate buffer solution at pH 7.1 (immobilization percentage: 87%). The immobilized enzyme exhibited activity on BAlME of 36 ⁇ moles/mg.hr at pH 7.1 and 35° C., whereby only L-isomer was found to be reactive.
• BAlME N-benzoyl-DL-alanine methyl ester
• the immobilized enzyme was washed with 5 M aqueous NaCl solution and 0.5 M phosphate buffer solution to elute and recover the enzyme. Recovery was 86% of the immobilized enzyme.
• the activity of the recovered enzyme in the state of a solution on BAEE was measured at 30° C. and pH 8.0 to be 68 ⁇ moles/mg.hr and on BAlME at 35° C. and pH 7.1 to be 37 ⁇ moles/mg.hr.
• the filtrate and washings were recovered and, from the ultra-violet adsorption strength at 280 nm of the recovered solution, the amount of the enzyme immobilized by adsorption on the resin was calculated to be 86 mg.
• the specific activity of this immobilized enzyme was measured using BAEE as substrate with pH stat (Hiranuma precision pH stat PS-11) at pH 7.0 and 30° C. under the condition of excess substrate over enzyme to be 3.8 ⁇ moles/mg.min (specific activity of liquid enzyme under the same measurement conditions being 9.0 ⁇ moles/mg.min). Accordingly, the total activity of the immobilized enzyme was 326 ⁇ moles/min. This immobilized enzyme maintained 88% of the activity after 10 days after immobilization.
• the enzyme was immobilized by adsorption on the resin.
• the amount of the immobilized enzyme was calculated from the ultra-violet absorption strength of the protein in the recovered liquid to be 92 mg.
• the activity of the immobilized enzyme when measured under the turn-over conditions at 40° C. and pH 7.0 using BAEE as substrate, was 4.1 ⁇ moles/mg.min which was 82% of that of the liquid state enzyme before immobilization.
• the amount of unaltered esters in the effluent was analyzed quantitatively to find that 100% thereof was hydrolyzed.
• the residual activity of the immobilized enzyme column after containued use for 10 days was as much as 80%.
• Immobilization was effected by the same procedure as in Example 7 by varying the resins as shown in the Table below. Measurements of activities were conducted under the same conditions as in Example 7 to give the results as listed in the same Table.
• Example 10 there was used the enzyme obtained similarly as in Example 8; in other Examples, the same commercially available Pronase E as in Example 7 was used.
• the specific surface area of each resin was measured, after vacuum drying each resin with heating at 60° C., by BET method by means of gas adsorption type surface area measuring instrument.
• the result obtained by immobilizing trypsin on a gel-type ion-exchange resin is also shown in the same Table.

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Citations (3)

• 1977
  • 1977-05-24 GB GB21936/77A patent/GB1536822A/en not_active Expired
  • 1977-05-27 US US05/801,246 patent/US4146432A/en not_active Expired - Lifetime
  • 1977-05-27 DE DE19772724081 patent/DE2724081A1/de not_active Withdrawn
  • 1977-05-31 FR FR7716516A patent/FR2353563A1/fr active Granted
  • 1977-05-31 NL NL7705966A patent/NL7705966A/xx not_active Application Discontinuation

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